A genetic defect underlies von Recklinghausen neurofibromatosis, known as type 1 neurofibromatosis (NF1), which is inherited as an autosomal dominant trait, and affects 1 in 3,500 humans. Analysis of the steps that lead to disease manifestations and development of therapeutic strategies against NF1 is complicated by the number of abnormalities in NF1 patients and the diversity of cell types involved. A National Institute of Health consensus panel has defined diagnostic criteria, two or more of which define a diagnosis of NF1. The criteria are: 1) six or more cafe-au-lait macules over 5 mm in greatest diameter (prepubertal) or over 15 mm (postpubertal); 2) freckling in the axillary or inguinal regions; 3) optic glioma; 4) two or more iris Lisch nodules; 5) a distinct osseus lesion; 6) a first degree relative with NF1; and 7) two or more nerve sheath tumors (neurofibromas) of any type or one plexiform neurofibroma. More recently it has become clear that up to 40% of children with NF1 show learning disabilities.
The NF1 gene is located on the proximal long arm of human chromosome 17, and extends about 320 kb of genomic DNA; all informative NF1 families showed linkage to this locus. Mutational analysis identified mutations in about 15% of patient DNA with no identified hot spots; remaining mutations may be in untested intron sequences. Mutations including translocations, insertions, deletions and base-pair substitutions predict truncated or inactive protein, with no evidence for dominant negative effects. In malignant tumors (neurofibrosarcomas, pheochromocytomas and juvenile myelocytic leukemias) associated with NF1, mutations in the previously normal somatic allele have been detected, suggesting that the NF1 gene can be categorized with tumor suppresser genes such a Rb and NF2. However, it remains unclear if mutations in both NF1 alleles is a prerequisite for the formation of benign lesions, including neurofibromas.
Ras is an intracellular messenger activated by binding GTP subsequent to receptor activation; Ras activation recruits the serine-threonine kinase raf to the plasma membrane and leads to activation of raf by an unknown mechanism. Raf activation leads to activation of a cascade of protein kinases (ERKs and MAPKs) and changes in gene transcription. Other pathways are also downstream of ras activation. In many tumor cells, mutant oncogenic alleles of ras genes specify structurally altered forms of Ras which have a reduced ability to hydrolyze GTP when compared with the wild type ras gene product (Gibbs et al., Proc. Natl. Acad. Sci. USA, 81:5704-5708 (1984); McGrath et al., Nature, 310:644-649 (1984); Sweet et al., Nature 311:273-275 (1984)). Additionally, these oncogenic forms of Ras have been found to exhibit a greatly diminished sensitivity to the ras GTPase activating protein (GAP) (Trahey and McCormick, Science, 242:1697-1700 (1987); Vogel et al., Nature, 335:90-03 (1988)). These findings suggest that the GTP-bound state of Ras represents an activated, signal-emitting form of Ras which is normally inactivated through GTP hydrolysis, yielding an inactive GDP-bound form of the protein. Because of their reduced intrinsic GTPase activity and resistance to GAP, oncogenic forms of Ras may be trapped in this activated state for extended periods of time, thereby flooding the cell with growth-stimulatory signals.
In part because Ras has no other apparent catalytic activities associated with it, it is hypothesized that Ras acts as a regulatory subunit of another protein that serves as its effector, releasing mitogenic signals when prompted to do so by activated, GTP-bound Ras. One candidate for such an effector protein is GAP itself. However, GAP has no obvious catalytic domains that might play a role in metabolic processes associated with cell proliferation. For this reason, GAP itself might act as a signal transducer which passes Ras-initiated signals on to bona fide effectors still further downstream in the signaling cascade.
The NF1 mRNA is &gt;12 kb in size, and encodes a 320 kd protein, neurofibromin. A 360 amino acid segment near the middle of the protein sequence shows 30% homology to the catalytic domain of the GTPase activating protein (GAP) and the yeast IRA1 and IRA2 proteins that stimulate GTPase activity of Ras proteins. The GAP-related domain of neurofibromin exhibits GAP activity toward human and yeast Ras proteins and complements the loss of function in yeast IRA mutants. Numerous studies have confirmed the GAP activity of full-length neurofibromin as well as the GAP-related domain. Loss of neurofibromin through mutations at the NF1 locus are predicted to cause a failure to terminate Ras signals, and to cause alterations in growth and differentiation of affected cells.
It has been shown that neurofibrosarcoma (malignant Schwann cell) cell lines contain little neurofibromin, and at the same time show increased levels of GTP bound to Ras, suggesting a correlation between loss of neurofibromin and Ras regulation in such cells (deClue et al., Cell, 69:265-273 (1992)). These data suggest that neurofibromin is the major regulator of Ras in malignant Schwann cells, in spite of the fact that these cells contain normal levels of p120 GAP protein. Decreasing levels of Ras-GTP in the neurofibrosarcoma cells by overexpressing p120 GAP led to reduced frequency of colony formation and decreased growth in agar, suggesting that Ras-GTP contributes to some of the transformed properties of these malignant Schwann cells.
Recently mice containing targeted inactivating mutations at the NF1 locus were studied (Brannan et al., Genes and Dev., 8:1019-1029 (1994)). Neurofibromin expression in adult mice and embryos from transgenic mice that were heterozygous or homozygous at the NF1 locus was analyzed. Neurofibromin was found to be present at roughly 50% of normal levels in heterozygous mice and was undetectable in homozygous mice, indicating that the latter were true nulls. Null embryos die between day 11.5 and 14 of gestation, prior to significant nerve development, apparently as a result of cardiac malformation.
Inhibition of farnesyl-protein transferase has been shown to block the growth of Ras-transformed cells in soft agar and to modify other aspects of their transformed phenotype. It has also been demonstrated that certain inhibitors of farnesyl-protein transferase selectively block the processing of the Ras oncoprotein intracellularly (N. E. Kohl et al., Science, 260:1934-1937 (1993) and G. L. James et al., Science, 260:1937-1942 (1993). Recently, it has been shown that an inhibitor of farnesyl-protein transferase blocks the growth of ras-dependent tumors in nude mice (N. E. Kohl et al., Proc. Natl. Acad. Sci U.S.A., 91:9141-9145 (1994).
Indirect inhibition of farnesyl-protein transferase in vivo has been demonstrated with lovastatin (Merck & Co., Rahway, N.J.) and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al., Science 245:379 (1989)). These drugs inhibit HMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids including farnesyl pyrophosphate. Farnesyl-protein transferase utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a farnesyl group (Reiss et al., Cell, 62:81-88 (1990); Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et al., Science, 249:1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci USA, 87:7541-7545 (1990)). Inhibition of farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in cultured cells. However, direct inhibition of farnesyl-protein transferase would be more specific and attended by fewer side effects than would occur with the required dose of a general inhibitor of isoprene biosynthesis.
Inhibitors of farnesyl-protein transferase (FPTase) have been described in two general classes. The first are competitive with farnesyl diphosphate (FPP) and can be structural analogs of FPP or not directly analogous. The second class of inhibitors are competitive with the protein substrates (e.g., Ras) for the enzyme. The protein substrate-competitive inhibitors that have been described are generally cysteine containing molecules that are related to the CAAX motif that is the signal for protein prenylation. (Schaber et al ibid; Reiss et. al., ibid; Reiss et al., PNAS, 88:732-736 (1991)). Such compounds may inhibit protein prenylation while serving as alternate substrates for the farnesyl-protein transferase enzyme, or may be purely competitive inhibitors (U.S. Pat. No. 5,141,851, University of Texas; N. E. Kohl et al., Science, 260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)). Recently, protein substrate-competitive inhibitors that lack a thiol moiety have been described (WO 95/09000; WO 95/09001; WO 95/10514; WO 95/10515; WO 95/10516; WO 95/08542; WO 95/11917; and WO 95/12612).
Inhibitors of FPTase have recently been described that incorporate characteristics of both farnesyl pyrophosphate and the CAAX motif (R. S. Bhide et al., Bioorg. Med. Chem. Lett., 4:2107-2112 (1994) and (V. Manne et al., Oncogene, 10:1763-1779 (1995)).
It is, therefore, an object of this invention to develop methods of treating and preventing benign proliferative disorder neurofibromatosis which utilize compounds which are known to be inhibitors of farnesyl-protein transferase.